There is widespread concern about growing resistance to antimicrobials, but hypochlorite, often referred to as bleach, has been the frontline chemical for a century due to its multiple mechanisms of microbial killing that has prevented widespread resistance. Bleach is used in household products, preventing spread of viruses like Ebola, and in commercial disinfection. For commercial applications especially, chlorinated organic compounds are often added to water to generate bleach in a metered fashion. One such application is the delivery of bleach for swimming pools and industrial water tanks via trichloroisocyanuric acid, commonly known as trichlor. Trichlor stabilizes bleach from light-catalyzed decomposition and so it is the standard mechanism for long-term protection of people in outdoor pools from the spread of infection due to viruses, bacteria and parasites like Giardia. For maintaining the disinfection, it is essential to remove cyanuric acid (CYA) when its concentration rises much above 1 mM (˜100 ppm) due to the equilibrium between cyanuric acid and hypochlorite that results in sequestration of most reactive chlorine species at high CYA concentrations.
Developing safe, sustainable disinfection and minimizing water use requires a mechanism to remove cyanuric acid when needed, but CYA is highly stable. While CYA is resistant to acid, base and temperatures of 200° C., some soil microbes easily degrade CYA enzymatically to liberate the three ring nitrogen atoms as ammonia that supports their growth in nitrogen-limiting environments. The microbial enzyme cyanuric acid hydrolase (CAH) hydrolyzes CYA with a kcat/KM>105 M−1 s−1. The use of CAH to remove CYA in pools and other disinfection waters has been considered (U.S. Pat. No. 8,367,389); however, one issue hindering the development of a robust system is the sensitivity of cyanuric acid hydrolase to hypochlorite. Hypochlorite is added to pools directly as sodium, lithium, or calcium hypochlorite. It also forms from the hydrolysis of trichlor that is added to pools. So, when cyanuric acid is present, hypochlorite is invariably present. Unfortunately, engineering a hypochlorite resistant biological material is not straightforward.
Accordingly, new compositions and methods for removing CYA from pools and other disinfection waters are needed. In particular, new CAH enzymes (e.g., thermostable) that are resistant to hypochlorite are needed.